Scanvac coolsafe laboratory freeze dryer

The preparation of LYS NPs was made according to 2³ full factorial design; therefore 8 samples were prepared. Namely, 0.6 g of lyophilized enzyme was dissolved in purified water to obtain 20 g of homogenous aqueous solution, and then each sample was mixed with 4ml of 2M Na₂SO₄ solution by using a magnetic stirrer for 1 hour. The 8 samples were centrifuged at 5000 rpm for 15 minutes by using a Hermle Z323K high performance refrigerated centrifuge (Hermle AG, Gossheim, Germany). Upon redispersion the total amount of precipitated LYS aqueous alginate solutions (25 ml) of conc. 0.004 and 0.006%w/v and pH 6 and 10 were added to each sample according to the factorial design. The samples were mixed with a high shear mixer (Ultra-Turrax, Germany) for 15 seconds, followed by mixing for 1 h and 2 h. After the mixing time, the samples were centrifuged again, the supernatant was separated and finally lyophilized at −25°C for 24 h under a pressure of 1.3 Pa, and then kept at 25°C for 24 h for secondary drying to obtain lyophilized powders by using a Scanvac Coolsafe laboratory freeze-dryer (LaboGene, Denmark). The samples were pre-frozen at -10±2°C before lyophilization. The samples were stored at -10±2°C until further investigations.

Insulin SLNs were obtained following a previously reported double-emulsion solvent-evaporation technique with some modifications [71 ]. The double emulsion (W1/O/W2) was prepared according to the following steps. First, the primary W1/O emulsion was prepared by adding 0.35 mL of insulin solution in 0.1 M aqueous HCl solution (2 mg/mL) dropwise to a phosphatidylcholine solution in cyclohexane (9 mg/mL), using an ultrasonic homogenizer (Hielscher, Germany) (0.5 cycles with 75% amplitude) for 1 min. Then, the resultant emulsion was added dropwise into 1.6 mL of 2% w/v poloxamer 188 aqueous solution (W2), using the homogenizing mixer (0.5 cycles with 75% amplitude) for another 1 min. The final mixture was left overnight in a laminar flow hood under stirring using a magnetic stirrer at 500 rpm, in order to allow for the evaporation of the organic solvent, thus forming the SLNs. Finally, freeze-drying was applied in the presence of 5% w/v trehalose as a cryoprotectant to obtain a lyophilized powder. For that purpose, a Scanvac CoolSafe laboratory freeze-dryer (Labogene, Lynge, Denmark) was operated at −40 °C for 12 h under a 0.013 mbar pressure, with an additional 3 h of secondary drying at 25 °C. The lyophilized powder was stored at 5 ± 3 °C until further investigation.

The procedure to prepare the HIP complex was adopted from a number of previous reports with some modifications based on our preliminary study [5 (link),7 (link),11 (link)]. LYZ (in a concentration of 4 mg/mL) and SDS (in concentration according to the finding of optimal molar ratio determination) were separately dissolved in phosphate buffer (pH 6.8) and the pH of the solutions was adjusted to the required value (e.g., 4, 6, 8 or 10) using 1 M HCl or 1 M KOH to ensure that the complexation takes place at the required pH value. Then, equal volumes of both solutions were added into Eppendorf tubes, mixed by pipetting, and allowed to stand for 30 min. Consequently, the final peptide concentration of 2 mg/mL was used in all experiments. After that, the samples were centrifuged at 15,000 rpm for 15 min at room temperature. The supernatant was removed, and the precipitated complex was dried.
For the samples dried by freeze drying, Scanvac Coolsafe laboratory freeze dryer (LaboGene, Lillerørd, Denmark) was used at −20 °C for 24 h under a pressure of 1.3 Pa and then kept at 25 °C for 24 h for secondary drying to obtain lyophilized powder [6 (link)].